Effect of High Temperature on Pseudomonas putida NBRI0987 Biofilm Formation and Expression of Stress Sigma Factor RpoS

Abstract

Pseudomonas is an efficient plant growth–promoting rhizobacteria; however, among the limiting factors for its commercialization, tolerance for high temperature is the most critical one. After screening 2,500 Pseudomnas sp. strains, a high temperature tolerant–strain Pseudomonas putida NBRI0987 was isolated from the drought-exposed rhizosphere of chickpea (Cicer arietinum L. cv. Radhey), which was grown under rain-fed conditions. P. putida NBRI0987 tolerated a temperature of 40°C for ≤ 5 days. To the best of our knowledge, this is the first report of a Pseudomnas sp. demonstrating survival estimated by counting viable cells under such a high temperature. P. putida NBRI0987 colony-forming unit (CFU)/ml on day 10 in both the absence and presence of MgSO4.7H2O (MgSO4) in combination with glycerol at 40°C were 0.0 and 1.7 × 1011, respectively. MgSO4 plus glycerol also enhanced the ability of P. putida NBRI0987 to tolerate high temperatures by inducing its ability to form biofilm. However, production of alginate was not critical for biofilm formation. The present study demonstrates overexpression of stress sigma factor σ
S
(RpoS) when P. putida NBRI0987 is grown under high-temperature stress at 40°C compared with 30°C. We present evidence, albeit indirect, that the adaptation of P. putida NBRI0987 to high temperatures is a complex multilevel regulatory process in which many different genes can be involved.

Full text

Effect of High Temperature on Pseudomonas putida NBRI0987
Biofilm Formation and Expression of Stress Sigma Factor RpoS
S. Srivastava Æ A. Yadav Æ K. Seem Æ S. Mishra Æ
V. Chaudhary Æ C. S. Nautiyal
Received: 13 September 2007 / Accepted: 26 November 2007 / Published online: 25 January 2008
Ó Springer Science+Business Media, LLC 2008
Abstract Pseudomonas is an efficient plant growth–
promoting rhizobacteria; however, among the limiting
factors for its commercialization, tolerance for high
temperature is the most critical one. After screening 2,500
Pseudomnas sp. strains, a high temperature tolerant–strain
Pseudomonas putida NBRI0987 was isolated from the
drought-exposed rhizosphere of chickpea (Cicer arietinum
L. cv. Radhey), which was grown under rain-fed condi-
tions. P. putida NBRI0987 tolerated a temperature of
40°C for B 5 days. To the best of our knowledge, this is
the first report of a Pseudomnas sp. demonstrating sur-
vival estimated by counting viable cells under such a high
temperature. P. putida NBRI0987 colony-forming unit
(CFU)/ml on day 10 in both the absence and presence of
MgSO
4
.7H
2
O (MgSO
4
) in combination with glycerol at
40°C were 0.0 and 1.7 9 10
11
, respectively. MgSO
4
plus
glycerol also enhanced the ability of P. putida NBRI0987
to tolerate high temperatures by inducing its ability to
form biofilm. However, production of alginate was not
critical for biofilm formation. The present study demon-
strates overexpression of stress sigma factor r
S
(RpoS)
when P. putida NBRI0987 is grown under high-temper-
ature stress at 40°C compared with 30°C. We present
evidence, albeit indirect, that the adaptation of P. putida
NBRI0987 to high temperatures is a complex multilevel
regulatory process in which many different genes can be
involved.
Introduction
Pseudomonas is an efficient plant growth–promoting rhi-
zobacteria, and certain isolates can enhance plant health [6–
8]. Pseudomonas can be found in many different environ-
ments, including soil, water, plant, animal, and human. To
persist successfully in a changing environment a microor-
ganism must sense such change and react appropriately.
Among the limiting factors for its commercialization, tol-
erance for high temperature is the most critical, resulting in
limited use because of its short shelf life [6, 12]. An
understanding of the growth of Pseudomonas isolated from
stressed conditions is likely only when the physiology of
these organisms has been carefully studied under these
suboptimal conditions. Moreover, Pseudomonas spp. with
the genetic potential for increased tolerance to these adverse
environmental stresses could enhance production of food in
semiarid and arid regions of the world [6, 9].
In view of the sensitivity of Pseudomonas spp. to the
high temperatures frequently encountered in the tropics and
subtropics, an investigation was conducted to find a means
by which to better protect or enhance the stress tolerance of
Pseudomonas spp. A high temperature–tolerant P. putida
NBRI0987 strain was isolated from the drought-exposed
rhizosphere of chickpea, which was grown under rain-fed
conditions. The goal of this study was to elucidate the
phenotypic and genetic attributes of high temperature–
tolerant P. putida NBRI0987 involved in enhanced biofilm
formation and protecting Pseudomonas from high
temperature.
S. Srivastava  A. Yadav  K. Seem  S. Mishra 
V. Chaudhary  C. S. Nautiyal (&)
Division of Plant Microbe Interactions, National Botanical
Research Institute, Rana Pratap Marg, Lucknow 226001, India
e-mail: csn@nbri.res.in
123
Curr Microbiol (2008) 56:453–457
DOI 10.1007/s00284-008-9105-0

Materials and Methods
Bacterial Strain, Culture Media, and Growth Conditions
Bacterial strains were isolated from the roots of field-
grown chickpea (Cicer arietinum L. cv. Radhey) in a
rain-fed area of Dholpur, Rajasthan, India, located at
latitude 26°42
0
north, longitude 77°54
0
east. High tem-
perature–tolerant rhizosphere–competent P. putida
NBRI0987 was isolated from field-grown chickpea (Cicer
arietinum L. cv. Radhey) rhizosphere as previously
described [6] and was identified using the Biolog system
(Biolog, Hayward, CA). Unless otherwise stated, P. put-
ida NBRI0987 was grown and maintained on nutrient
broth (NB) or nutrient agar (NA) (HI-MEDIA Laborato-
ries, Bombay, India).
High-temperature Stress and Quantification of Biofilm
Formation and Alginate Production
The stress tolerance of Pseudomonas strains toward
temperature was tested by growing them on NB broth in
150-ml Erlenmeyer flasks containing 50 ml NB with an
initial inoculation of approximately 1 9 10
7
CFU/ml as
previously described [8]. The flasks were incubated on a
New Brunswick Scientific (Edison, NJ) Innova model
4230 refrigerated incubator shaker at 180 rpm. Popula-
tions at each time point in the Figs. 1 through 3 represent
the means of three independent experiments. An SD
of ± 0.25 log CFU/ml was found for the viable cell
counts. The method for determining the extent of bacterial
adherence to the microtiter well surfaces has been
described elsewhere [10]. Briefly, the bacterial superna-
tants were discarded after incubation, and loosely
adherent bacteria were removed by three washes with
phosphate-buffered saline (pH 7.2). The microtiter plates
were then inverted and allowed to dry before each well
was filled with 25 ll 0.1% (w/v) crystal violet (CV)
solution and incubated at room temperature for 30 min-
utes. Unbound CV was removed by three washes with
water, and the plates were inverted to dry. Cell-bound CV
was released from bacterial cells by the addition of 200 ll
95% ethanol and, after incubation at room temperature for
30 minutes on a rotary shaker, the concentration of CV in
each solution was determined by the optical density
reading at 590 nm (Tecan Infinite 200 Microplate Reader,
Ma
¨
nnedorf, Switzerland). Similarly, wells containing only
NB but no bacteria were used as negative controls. Levels
of alginate were determined as previously described [17].
Bacterial population, bacterial adherence, and alginate
measurements represent the means of three independent
experiments.
Isolation of Total RNA and Reverse Transcriptase
Reaction–Polymerase Chain Reaction
P. putida NBRI0987 cells were grown in NB at 30°C and
40°C for 20 hours. One milliliter of the culture was taken to
prepare RNA. The cells were immediately frozen at -80°C.
Total RNA was isolated from the frozen cells using RNA-
Easy Mini Kit (Qiagen, Hilden, Germany) as described by
the manufacturer. Residual DNA was digested using DNase
(Qiagen) treatment. Thus, total RNA obtained was checked
on 1.5% formamide denaturing gel and quantified using a
spectrophotometer (Shimadzu, Kyoto, Japan). Using equal
amount of RNA (5 lg) from each sample, cDNA synthesis
was performed using the RevertAid H Minus First-Strand
cDNA Synthesis Kit (Fermentas UAB, Vilnius, Lithuania)
as described by the manufacturer.
Primer sequences used for 16S [2] and rpoS [5] were as
previously described. Primer sequences used for AlgT,
AlgD, and GreA were designed using gene sequences from
gene accession numbers GI:24982893 [5’-
Fig. 1 Effect of MgSO
4
.7H
2
O (MgSO
4
) (25 mM) plus glycerol (5%
v/v) supplementation on the growth of P. putida NBRI0987. Control,
MgSO
4
, glycerol, and glycerol plus MgSO
4
at (A)30°C and (B)40°C.
Bacterial survival was determined at the indicated times in triplicate,
and results are the means of three independent experiments.
SD ± 0.25 log CFU/ml was found for the viable cell counts.
Variation (SD) was within symbol dimensions
454 S. Srivastava et al.: High Temperature–Tolerant P. putida
123

gttgcaagcctgaacgatg-3’], GI:24982742 [5’-actgtctggagcct
ttgcat-3’], and GI:24986475 [5’-cgacatggaatacccacagg-3’],
respectively. All experiments were independently repeated
three times. Oligonucleotides used in this study were syn-
thesized by Bangalore Genei (Banglore, India). Polymerase
chain reaction amplification of the respective genes were
performed using equalized cDNA concentration (approxi-
mately 25 ng) from each RNA sample in a 20-ll reaction
mixture containing 2.0 ll Taq buffer (10 mM Tris HCl, pH
9.0, 50 mM KCl, 1.5 mM MgCl
2
, and 0.01% gelatin),
0.5 mM each of forward and reverse primers, deoxyribo-
nucleoside triphosphate (0.1 mM each), and 0.3 U Taq
polymerase from Bangalore Genei. After 3 minutes of initial
denaturation at 94°C, the reaction involved 30 cycles of 94°C
for 1 minute, 1 minute at annealing temperature 63°C for
rpoS and 60°C for rest of the genes, extension at 72°C for
1 minute, and final extension for 10 minutes at 72°Cina
Flexigene thermocycler (Techne, Cambridge, UK). Ampli-
fied products were loaded on 1.2% agarose gel, and the
molecular mass of the amplified DNA was estimated by
comparing with the 500-bp DNA ladder (Fermentas UAB,
Vilnius, Lithuania). Scanning the gels was performed on the
Gel-Documentation System (Uvitec, Cambridge, UK). All
the experiments were independently repeated three times.
Results and Discussion
After screening a total of [ 2,500 Pseudomnas sp. strains,
a high temperature–tolerant strain in P. putida NBRI0987
was isolated that tolerated a temperature of 40°C for B 5
days (Fig. 1). To the best of our knowledge, this is the first
report of a Pseudomnas sp. demonstrating survival esti-
mated by counting viable cells under such a high
temperature. We studied the effects of various carbon,
nitrogen, and metals alone and in various combinations on
the survival of P. putida NBRI0987 at 40°C (data not
provided). Survival of the strain was monitored at 30°C
(Fig. 1A) and 40°C (Fig. 1B), in the presence of
MgSO
4
.7H
2
O (MgSO
4
; 25 mM) plus glycerol (5% v/v)
for B 10 days. P. putida NBRI0987 survived in NB con-
taining MgSO
4
plus glycerol for B 10 days (Fig. 1).
Enhanced cell survival at 30°C (Fig. 1A) and 40°C
(Fig. 1B), was observed in the presence of MgSO
4
plus
glycerol for B 10 days compared with MgSO
4
and glyc-
erol used alone. P. putida NBRI0987 CFU/ml on day 10 in
the presence of 0, MgSO
4
, glycerol, and MgSO
4
plus
glycerol at 30°C were 1.7 9 10
9
, 1.3 9 10
11
, 1.3 9 10
11
and 1.7 9 10
13
/ml, respectively (Fig. 1A). At 40°C, P.
putida NBRI0987 CFU/ml on day 10 in the presence of 0,
MgSO
4
, glycerol, and MgSO
4
plus glycerol were 0,
1.2 9 10
9
, 8.5 9 10
8
and 1.7 9 10
11
, respectively (Fig. 1
B). In general, P. putida NBRI0987 efficiently tolerated
high temperature (40°C) in the presence of MgSO
4
plus
glycerol: CFU/ml of P. putida NBRI0987 were greater in
the presence of MgSO
4
plus glycerol compared with its
absence (Fig. 1).
Bacterial biofilms have a significant impact in medical,
industrial, and environmental settings. Numerous environ-
mental parameters influence whether biofilms are
successfully established in these settings, as was found for
P. fluorescens [10] and Sinorhizobium meliloti [11].
Because we noted enhanced survival of P. putida
NBRI0987 grown in the presence of NB supplemented
with MgSO
4
plus glycerol, and observation suggested that
biofilm formation may be involved because it represents a
survival strategy, we tested this possibility. The effect of
MgSO
4
plus glycerol was studied on the biofilm-formation
ability of P. putida NBRI0987 in the wells of the microtiter
plates at 30°C and 40°C. Fig. 2 shows that maximal biofilm
formation was observed 48 hours after incubation in the
microtiter plate wells in the presence of MgSO
4
plus
glycerol for up to 48 hours compared with their use sepa-
rately. The results indicate that the combination of MgSO
4
plus glycerol enhances the ability of P. putida NBRI0987
to tolerate high temperature by inducing it to a sessile
mode of life, i.e., a biofilm (Fig. 2).
The present study demonstrates that rpoS expression is
induced when P. putida NBRI0987 is grown under tem-
perature stress at 40°C compared with 30°C (Fig. 3). The
regulation and function of stress sigma factor r
S
(also
known as r
38
), encoded by rpoS, has been studied in a
variety of Gram-negative bacteria, especially in Esche-
richia coli and Pseudomonas spp. [3–5, 10]. Certain
functions of RpoS are similar in Pseudomonas spp. and in
Fig. 2 Effect of MgSO
4
.7H
2
O (MgSO
4
) (25 mM) plus glycerol (5%
v/v) supplementation on biofilm formation of NBRI0987. Control,
MgSO
4
, glycerol, and glycerol plus MgSO
4
(column A) at 24 hours at
30°C, (column B) at 48 hours at 30°C, (column C) at 24 hours at
40°C, and (column D) at 48 hours at 40°C. Values represent the
means of three independent experiments, and vertical bars indicate SE
S. Srivastava et al.: High Temperature–Tolerant P. putida 455
123

enteric bacteria. In particular, survival during osmotic,
heat, or oxidative stress is decreased in the rpoS mutants of
P. aeruginosa, P. putida, and P. fluorescens [12]. rpoS has
been reported as being an important factor for adaptation to
stress conditions because it produce alginate, which is
important for survival under stress conditions [5]. There-
fore, the affect of high temperature on alginate biosynthesis
pathway was also studied. In this study, we examined the
role of alginate production at 30°C and 40 °C and in the
presence and absence of MgSO
4
plus glycerol. Alginate
produced was 0.396 ± 0.035 lg/mg wet biomass at 30 °C
and 0.347 ± 0.067 lg/mg wet biomass at 40 °C after 20
hours of incubation. Although supplementation of MgSO
4
plus glycerol served as a stress reliever by supporting
biofilm formation and better survival, it decreased alginate
formation: The amount of alginate produced was
0.204 ± 0.07 at 30°C and 0.218 ± 0.03 at 40°C after
20 hours of incubation. Quantitative estimation of alginate
demonstrated that supplementation of MgSO
4
plus glycerol
relieves the stress imposed by high temperature and sup-
ports growth by forming more biofilm but not by the
synthesis of alginate. Our data is in accordance with earlier
findings that stressed environmental response results in
more alg D expression, but because Pseudomonas are
nonmucoid bacteria, they produce more or less equal
alginate under either condition, indicating that the pro-
duction of alginate is not critical for biofilm formation
[14, 17].
Alternative sigma factor (algT) was overexpressed,
followed by algD the structural gene of the alginate bio-
synthetic pathway, at 40°C compared with 30°C (Fig. 3).
The function of algT in the regulation of alginate synthesis
has been well documented [1, 15]. Results are in agreement
with previous reports stressing that tolerance ability is
imparted by the overexpression of algT, which in turn takes
over the charge of rpoS and thereby governs the synthesis
of alginate biosynthetic gene algD [15]. This results in
more biofilm formation, thus protecting cells from stress
produced by high temperatures. Overexpression of rpoS
and algT at 40°C was further supported by the overex-
pression of the greA family of transcription elongation
factor with regard to temperature tolerance in our study
(Fig. 3). greA is known to support the growth of E. coli at
high temperatures, and it has also been reported to be
induced by heat shock, salt shock, and oxidative stress in
Bacillus subtilis [13, 16]. Therefore, data are indicative of
the role of the transcription elongation factor greA in
conferring stress tolerance in P. putida NBRI0987 toward
high temperatures in accordance with rpoS. Our results
suggest that the adaptation of P. putida NBRI0987 to high
temperatures is a complex multilevel regulatory process in
which many different genes can be involved. Our group has
initiated molecular studies to isolate temperature-tolerant
defective mutants and to identify the genes from which
they derive, which will lead to a better understanding of the
mechanism of temperature tolerance in bacteria.
Acknowledgments Thanks are due to the director of the National
Botanical Research Institute, Lucknow, for necessary support of this
study. The study was supported by Task Force Grant No. SMM-002
from the Council of Scientific and Industrial Research, New Delhi,
India, awarded to C. S. N.
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